Anaplerotic reprogramming of endothelial cells in pulmonary hypertension.

项目摘要 肺动脉高压（PH）是一种肺血管细胞失控的致命疾病 增殖过度的内皮细胞（EC）生长导致丛状病变的形成， PH患者肺动脉内的特征性癌样变化。 PH患者肺部的氧化应激导致超氧化物与NO反应。 形成高活性过氧亚硝酸根（ONOO-），进而产生硝基酪氨酸 蛋白质的修饰。PH患者的肺组织蛋白质硝化水平增加。 我们最近发表的工作表明，Akt中酪氨酸350残基的硝化导致了Akt的蛋白质合成。 持续激活Akt信号传导。Akt的激活是细胞存活和应激的良好描述。 但是Akt的激活受到调节激酶/磷酸酶（PTEN，PI 3 K， PDK 1和mTOR），其控制Akt活性并确保其仅暂时活跃。病理 Akt途径的激活与多种癌症有关。我们假设在PH中， Akt的硝化作用增加eNOS向线粒体的转运， 磷酸化并激活丙酮酸羧化酶（PC），其上调回补。这 回补的激活然后导致内皮细胞（EC）的病理性过度增殖， 此外，我们建议使用Akt-1靶向病理性Akt信号传导， 结合肽与抗氧化剂（屏蔽肽）偶联，仅影响硝化介导的Akt activation.我们将测试这些假设在以下目标：目的1：阐明硝化的作用 （Y350）介导的Akt激活在EC回补激活中的作用。目的2：确定是否抑制 Akt硝化作用破坏了细胞凋亡抗性和增殖性EC表型的形成。目标3： 在SU 5416/缺氧PH模型中检测Akt屏蔽肽对EC过度增殖的影响。

PROJECT SUMMARY Pulmonary arterial hypertension (PH) is a fatal disease with uncontrolled pulmonary vascular cell proliferation. Excessive endothelial cell (EC) growth leads to the formation of plexiform lesions, which are a characteristic cancer-like change within pulmonary arteries of patients with PH. The increased oxidative stress in lungs of patients with PH leads to the reaction of superoxide with NO. This results in the formation of highly reactive peroxynitrite (ONOO-) which, in turn, can produce nitro-tyrosine modifications in proteins. Lung tissues from patients with PH have an increased level of protein nitration. Our recently published work indicates that the nitration of tyrosine 350 residue in Akt leads to the sustained activation of Akt signaling. The activation of Akt is a well described cell survival and stress response but Akt activation is tightly regulated by the regulatory kinases/phosphatases (PTEN, PI3K, PDK1 and mTOR) that control Akt activity and ensuring that it is only temporarily active. Pathological activation of the Akt pathway has been implicated in various cancers. We hypothesize that in PH, nitration of Akt increases translocation of eNOS to mitochondria, which downregulates oxidative phosphorylation and activates Pyruvate Carboxylase (PC), which upregulates anaplerosis. This activation of anaplerosis then leads to pathological hyper-proliferation of endothelial cells (EC) and plexiform lesion formation in PH. Further, we propose to target pathological Akt signaling using an Akt- binding peptide conjugated with an antioxidant (shielding peptide) that only affects nitration mediated Akt activation. We will test these hypotheses in the following Aims: AIM 1: To elucidate the role of nitration (Y350) mediated Akt activation in activation of anaplerosis in EC. AIM 2: To determine whether inhibition of Akt nitration disrupts formation of the apoptosis resistant and proliferative EC phenotype. AIM 3: To examine the effect of Akt shielding peptide on EC hyper-proliferation in the SU5416/hypoxia PH models.